Normal enteric bacteria have been shown to seed inflammatory foci in the peritoneal cavity by direct contamination through anatomically intact bowel, thereby producing or perpetuating bacterial peritonitis. Prior work in this grant has shown that fluorescent latex beads as well as bacteria will pass through the bowel wall to the serosa. We have provided evidence that particulate transport takes place within macrophages which normally reside in the submucosa of the bowel. We now plan to employ isolated, perfused, mucosal/submucosal membranes in vitro to investigate by what mechanisms inert particles (latex beads) or bacteria (E. coli) pass through the intact epithelial layers of the alimentary tract to reach the submucosa. The bowel wall will be split in a plane between the submucosa and the circular muscle. The mucosal/submucosal membrane will be mounted in Ussing chambers permitting both sides of the membrane to be oxygenated and perfused. A stable potential difference indicates viability and a stable resistance indicates electrochemical impermeability of the membrane. Labelled beads or bacteria introduced on the mucosal surface pass through the epithelial membrane to the submucosal surface where they can be quantitated by fluorescence and/or quantitative bacteriology. Preliminary kinetic and microanatomical studies suggest that particulate passage is an energy-dependent function of the living intestinal epithelial cell which incorporates the particle and passes it through to the lamina propria. The proposed studies are designed to investigate the microanatomical and functional details of this normal mechanism which will be compared with gut from animals with infection and shock. Furthermore, the importance of enteropathogenic virulence characteristics and bacterial adherence will be assessed. The role of intestinal mucus, tight junctions, brush border glycocalyx, lamina propria, T cells, various cytokines, and IgA on normal transepithelial passage of bacteria will be tested. Selected probes to evaluate the role of intraepithelial pinocytosis, phagocytosis, and bacterial killing will be employed. Considerable information should be gained on the mechanism by which the intestinal flora gain entrance into the body under normal conditions and during physiologic stress.